Multi-tiered approach to detect autoimmune cross-reactivity of therapeutic T cell receptors

T cell receptor (TCR)–engineered T cell therapy using high-affinity TCRs is a promising treatment modality for cancer. Discovery of high-affinity TCRs especially against self-antigens can require approaches that circumvent central tolerance, which may increase the risk of cross-reactivity. Despite the potential for toxicity, no standardized approach to screen cross-reactivity has been established in the context of preclinical safety evaluation. Here, we describe a practical framework to prospectively detect clinically prohibitive cross-reactivity of therapeutic TCR candidates. Cross-reactivity screening consisted of multifaceted series of assays including assessment of p-MHC tetramer binding, cell line recognition, and reactivity against candidate peptide libraries. Peptide libraries were generated using conventional contact residue motif–guided search, amino acid substitution matrix–based search unguided by motif information, and combinatorial peptide library scan–guided search. We demonstrate the additive nature of a layered approach, which efficiently identifies unsafe cross-reactivity including one undetected by conventional motif-guided search. These findings have important implications for the safe development of TCR-based therapies.

peptide CD20p188-196 along with poly(I:C) and anti-mouse CD40 monoclonal antibody (Supplementary Figure S1A). Female mice between 6 and 10 weeks of age were intraperitoneally injected with a mixture of the CD20p188-196 peptide 100 µg (>95% purity, Peptide 2.0, Inc and Genscript), poly(I:C) 50 µg (InvivoGen), and anti-CD40 monoclonal antibody (clone FGK4. 5,BioXcell) in PBS adjusted to a final total volume of 200 µL per mouse on days 0, 14, and day 28 (anti-CD40 monoclonal antibody was omitted from day 28). Peripheral blood was collected on days 7, 21, and 35 to assess for circulating tetramerbinding T cell frequencies. Chronological changes in T cell responses were assessed by obtaining peripheral blood from each mouse between scheduled vaccinations. Additionally, CD20 tetramers+ CD8+ T cells were isolated from B6.HLA-A2 mice with homozygous deletion of autoimmune regulator (Aire) (B6.HLA-A2-AireKO) using the same vaccination method. The Aire gene encodes a protein that regulates tissue-specific antigen expression in medullary thymic epithelial cells (63), playing a critical role in central tolerance. Therefore, Aire deficient mice are assumed to have a different underlying TCR repertoire compared to Aire competent mice, although murine CD20 or the mouse proteome do not contain the intended epitope sequence from human CD20.

Murine T-cell in vitro T-cell stimulation
Following vaccinations, mice were humanely euthanized and lymphoid tissues were harvested on day 35. Red blood cells were lysed using ACK Lysing Buffer (Lonza) and single cell suspension was filtered through sterile 40 µm filter. Cells were used for in vitro T-cell simulation: Cells were re-suspended in RPMI 1640-based media (as described above) with an addition of 2-mercaptoethanol 50 μM, recombinant human IL-2 (rh-IL-2) 30 IU/mL, and the CD20p188-196 peptide 1 µM. Cells were plated in tissue-culture treated 24-well plate, 6e6 cells/well in 2 mL volume, and cultured for 1 week in 37°C. On day 42 (i.e. a week after in vitro stimulation) cells were re-analyzed on flow cytometry or used in co-culture assays. Mouse splenocytes were incubated with HLA-A*02:01-CD20p188-196 tetramer conjugated with PE (1 µL of p-MHC tetramer per 1e6 T cells in 10 µL MACS buffer) on ice for 30 minutes protected from light. Then, Anti-PE-MicroBeads (Miltenyi) was used to perform positive selection of PE+ population following the manufacture's instruction.
T cells bound to CD20 tetramers were magnetically isolated from mouse splenocytes (either immediately following tissue harvest or after in vitro stimulation), and the paired TCR α chain and β chain nucleotide sequences at a single cell level were determined using the 10x Genomics platform.

Isolation of EBV LMP2-specific TCR from human PBMC
From HLA-A*02:01+ human PBMC, monocyte-derived dendritic cells were generated using adherence technique. Briefly, PBMC were resuspended in CellGenix GMP DC medum (Cellgenix) with 1% heatinactivated human AB serum (HS) (Gemini Bio) in 6-well TC-treated plate at 1 e7 cells/well. Following 90 minutes of incubation in 37°C, non-adherent cells were gently removed by swirling and pipetting with warm serum-free media. Cells adherent on the bottom of the plate representing monocytes were cultured for overnight with Cellgenix DC media, 1% HS with IL-4 1000 IU/mL and GM-CSF 800 IU/mL to make immature dendritic cells (DCs). The next morning, following cytokines were added to the culture to achieve final concentrations of TNFα 10 ng/mL, IL-1β 10 ng/mL, IL-6 1000 IU/mL, and PGE2 1 µg/mL. Approximately 24 hours later, matured DC were harvested and loaded with minimal epitope peptide (CLGGLLTMV) at 1 µM for 30-60 minutes in 37°C, irradiated with 40 Gy, and co-cultured with autologous CD8+ T cells (autologous to the DC) at an E:T = 10:1, CD8+ 1e6 cells and DC 1e5 cells/well in 48-well TC-treated plate, in RPMI-based media (RPMI, 10% HS, L-alanyl-L-glutamine dipeptide and penicillin / streptomycin 100 μg/mL) in the presence of IL-21 30 ng/mL. Three days later, IL-7 10 ng/mL, IL-2 25 IU/mL, and IL-15 2 ng/mL were added in the co-culture media. Following 7-14 days of co-culture, the presence of EBV LMP2-tetramer binding CD8+ T cells were assessed with flow cytometry analysis, and tetramer binding cells were isolated and used for TCR sequencing. In vitro stimulation of CD8+ T cells with autologous PBMC were repeated as necessary.

Single cell TCR paired sequencing
Single cell TCR alpha/beta paired sequencing and all analyses were performed at the National Cancer Institute Center for Cancer Research Single Cell Analysis Facility. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov). From the samples that yielded productive V(D)J spanning pairs with clonotype enrichment, 10 unique TCRs were identified (Supplementary Table  S1).
Single Cell -Partitioning and Library Preparation: Single cell suspensions of T cells stored in RNA Protect (Qiagen) were aliquoted 25ul each into several microcentrifuge tubes and volumed up to 1mL with PBS. These were centrifuged at 300g for 5mins, supernatant removed, and then cells were resuspended in approximately 50uL of remaining buffer volume. Cell counts were measured using a fluorescent cell counter with propidium iodide and acridine orange (LunaFL, Logos Biosystems). Cell suspensions were loaded at maximum volume onto the 10x Genomics Chromium platform using the 5' v1.1 immune profiling chemistry (10x Genomics). Following partitions, reverse transcription, and cDNA amplification, mouse TCR enrichment libraries were prepared according to vendor recommendations.
Single Cell -Sequencing: Sequencing of single cell mouse TCR libraries were performed on an Illumina NextSeq 550/500 instrument with paired end 150bp reads and an 8bp read for sample indices. Samples were multiplexed for sequencing and reads were combined from multiple sequencing runs to achieve at least 10,000 reads per cell on average for TCR libraries for all samples.
Single Cell -Data Processing: Data was processed using the 10x Genomics cellranger pipeline (v3.1.0) to demultiplex reads and then align reads to a GRCm38 VDJ reference for single cell TCR data (refdatacellranger-vdj-GRCm38-alts-ensembl-3.1.0). UMI-adjusted aligned reads were used to generate a TCR clonotype matrix that was used for downstream analysis.

CDR3 motif analysis
An R package "ggseqlogo" was used to generate position weight matrix of CDR3 amino acid sequences (64). The '-' character was used as a place holder to align CDR3 amino acid sequences of different lengths across multiple TCR clonotypes.

Gamma-retroviral supernatant production
Gamma-retroviral supernatant production was performed by co-transfection of 293GP cells with a plasmid encoding the envelope RD114 and the retroviral (MSGV) TCR expression vectors using the lipofectamine 3000 (Life Technologies) following the manufacturer's protocol. Viral supernatant was collected 48, 72 and 96 hours after the transfection and frozen and stored in -80°C until use.

Incucyte
Confluency of transduced T cells were monitored with Incucyte S3 (Essen BioScience, Sartorius). Five days after the transduction and the confirmation of transduction efficiency, TCR-transduced T cells were counted and plated at 2.5 e5 cells/well in 2 mL volume in TC-treated 24-well plate. Phase contrast images were taken, and confluency was calculated using the Incucyte software (Incucyte 2022B Rev1) following the manufacture's protocol.
In silico search using the BLAST Protein BLAST was accessed on April 12, 2021, May 1, 2021, November 28, 2022, and December 14, 2022 (70,71). Query sequence was the CD20p188-196 peptide sequence (SIFLGILSV) and the search database was set as "Non-redundant protein sequences (nr)" with a filter for homo sapiens (taxid:9606). We chose the "blastp" algorithm and set the algorithm parameters as follows: max target sequences of 1000, check the box for "automatically adjust parameters for short input sequences" which led the blastp program to automatically use PAM30 matrix, gapcosts of 9/1, word size 2, and expect value of 200,000.

Supplementary Figure S2. Fratricide of TCR-transdcued T cells:
Transduced T cells were plated in 24-well TC-treated plate at the initial seading desity of 2.5e5 cells/well. EBV LMP2 TCR T cells were co-cultured with K562A2 cells loaded either with the wild-type EBV LMP2p426-434 peptide or with peptides with alanine substitution on each residue at 1 µM. IFNγ levels in the overnight co-culture supernatant was measured with ELISA. Second and nineth residues are HLA-A*02:01 anchor residues. Based on the alanine scanning result, residues on positions 4, 5, 6, and 7 were inferred to be the non-anchor residues important for TCR recognition. Contact motif-guided in silico search rule in ScanProsite was x-x-x-G-L-L-T-x-x. Figure S7. TCRs recognizing HLA-A*02:01-restricted epitope of CD20 derived from mice and humans share TCRβ motif: